The invention pertains to a procedure for the fusing of cells in which the cells in a cell suspension in the fusion space are brought close together by external forces and disruptions are caused by external effects in the membrane structure of the adjacent cells; these disruptions lead to the formation of membrane bridges between adjacent cells and to the fusion of the cells.
If two cells in a suspension touch one another and close contact develops between the membranes of the two cells, they should fuse since the components in the membranes are mobile. Such spontaneous fusion of cells is observed extremely rarely or not at all under natural conditions. A known exception is the fertilization of an egg cell by a semen cell during sexual reproduction. Spontaneous fusion is hindered by the negative charge of the phospholipids and other membrane components. This negative charge causes the cells to repel once they have come to within a certain distance of one another. Cell fusion requires, however, that the two membranes be able to come within less than 10.sup.-7 cm.
The fusion of cells which is carried out by technical means can be used in a broad range of applications. Thus, for biomedical research it is of great interest to fuse a large number of cells. When the cells, which are created by fusing several or, if necessary, many cells (for example, 1,000 to 10,000 corpuscles), are of the appropriate size, microelectrodes micro pressure-measuring probes and other sensors can be introduced into the large cell without irreversibly destroying the membrane. The technique of directly recording a number of cells and membrane functions by means of the sensors is of importance to clinical diagnosis, for instance in the early detection of diseases as well as in general for basic research.
The cell fusion technique can also be used to form hybrid cells by fusing two cells of different origin. In this case cell hybrids can be formed from plant cells from which, in turn, whole plants can be cultivated or cell hybrids can be obtained from animal cells by means of which monoclonal antibodies, for instance, antibodies to tumors and leukemic cells, can be produced. As an example, let us mention the fusion of a lymphocyte cell with a myeloma cell, which is of great interest particularly to medicine and pharmacology. Certain lymphocytes form antibodies to foreign substances in the organism, for instance, to a foreign protein which has been injected into the blood stream. If the lymphocytes are isolated and fused with a tumor cell such as a myeloma cell, then there is the chance that a so-called hybridoma cell will form which will have the properties of both parent cells. This cell will produce antibodies which are specific only to the corresponding foreign substance (so-called monoclonal antibodies). The cell will not die and, in contrast to a normal differentiated cell such as the lymphocyte, it can be permanently reproduced in nutrient media.
A procedure for the fusion of the cells of the type mentioned in the introduction is known from Biochemica et Biophysica Acta, 694 (1982), 227-277 (Electric Field-Medicated Fusion and Related Electrical Phenomena, U. Zimmermann). In the case of this known procedure (the course of which can be observed under a microscope), the membrane contact is generated between at least two cells by applying an alternating, slightly nonhomogeneous field. Because of polarization processes in the cell, the electric field creates dipoles which mutually attract if the cells come close to one another during their migration in the electrical field (so-called dielectrophoresis). After a row of cells has been formed, disruptions in the membrane structures between adjacent cells are triggered by an electric breakdown pulse (J. Membrane Biol. 67, 165-182 (1982), Electric Field-Induced Cell-to-Cell Fusion, U. Zimmermann and J. Vienken). According to previous model concepts, holes are created in the membrane contact zone of adjacent cells in the process which lead to a cytoplasmic continuum between the two cells and to the formation of lipid bridges between the membranes of the adjacent cells. The lipid molecules are no longer arranged in their original membrane. As soon as a bridge has formed, energy factors lead to rounding off of the formation which has developed and which consists of cells which are connected by means of the lipid bridges.
In the implementation of the known procedure for collecting cells by dielectrophoresis, however, it is necessary for the solution which holds the cells during the execution of the procedure to be as non-conductive as possible, otherwise the generation of heat will be excessive, and this will lead to turbulences and disruption of the close membrane contact between adjacent cells. This is disadvantageous to the extent that the cells have little or no tolerance for a solution which is only slightly conductive and therefore the cells can suffer damage in a medium of low conductivity, and this impairs, among other things, their longevity.
A way is also known of using certain chemicals such as polyethylene glycol (PEG) or inactivated viruses to eliminate the electrical repelling forces which occur when there is a short distance between the membranes of two cells. Both viruses and PEG interlace the two cell membranes so that close membrane contact is created. At the same time, the viruses and PEG create disruptions in the membrane structure which can be reinforced by establishing non-physiological conditions such as adding high concentrations of calcium ions and selecting a very high or low pH value. The effect of these disruptions is that holes form in the membrane contact zone and this leads to the formation of phospholipid bridges between the adjacent cell membranes. This leads to the fusion of the two cells, forming a new unit.
In the implementation of the known procedures mentioned above, however, the number of cells to be fused cannot be controlled. On the one hand, an excessively low cell density in the solution containing the cells leads in practice to no fusion products since the cells do not come into contact with one another. On the other hand, a cell density which is sufficient to achieve fusion (and which can also be achieved by centrifuging the solution containing the cells) in an uncontrolled fashion leads to products which consist of doubles, triples, quadruples or multiples of individual cells.
In particular with regard to the formation of hybrid cells by fusing only two cells of different origin, the above-mentioned known procedures are very unsatisfactory. The reason is that cell hybrids which feature a new combination of properties must be isolated through the use of very time-consuming selection methods after the known procedures are implemented. The production of cell hybrids from plant cells from which, in turn, whole plants can be cultivated or of cell hybrids from animal cells by means of which monoclonal antibodies, for instance to tumors and leukemia can be obtained, requires a fusion technique in which only two cells are fused at a time.
It is therefore a principal object of the present invention to create a procedure for fusing cells during which the cells can be in a more conductive solution but the cells are still initially brought close together by the actual procedural step of the fusion itself.